Interpreting the biological relevance of bioinformatic analyses with T-DNA sequence for protein allergenicity.

Global regulatory agencies require bioinformatic sequence analysis as part of their safety evaluation for transgenic crops. Analysis typically focuses on encoded proteins and adjacent endogenous flanking sequences. Recently, regulatory expectations have expanded to include all reading frames of the inserted DNA. The intent is to provide biologically relevant results that can be used in the overall assessment of safety. This paper evaluates the relevance of assessing the allergenic potential of all DNA reading frames found in common food genes using methods considered for the analysis of T-DNA sequences used in transgenic crops. FASTA and BLASTX algorithms were used to compare genes from maize, rice, soybean, cucumber, melon, watermelon, and tomato using international regulatory guidance. Results show that BLASTX for maize yielded 7254 alignments that exceeded allergen similarity thresholds and 210,772 alignments that matched eight or more consecutive amino acids with an allergen; other crops produced similar results. This analysis suggests that each nontransgenic crop has a much greater potential for allergenic risk than what has been observed clinically. We demonstrate that a meaningful safety assessment is unlikely to be provided by using methods with inherently high frequencies of false positive alignments when broadly applied to all reading frames of DNA sequence.

Impact of Food Matrices on Digestibility of Allergens and Poorly Allergenic Homologs.

Background: Protease resistance is considered a risk factor for allergenicity of proteins, although the correlation is low. It is nonetheless a part of the weight-of-evidence approach, proposed by Codex, for assessing the allergenicity risk of novel food proteins. Susceptibility of proteins to pepsin is commonly tested with purified protein in solution. Objective: Food proteins are rarely consumed in purified form. Our aim was to evaluate the impact of experimental and endogenous food matrices on protease susceptibility of homologous protein pairs with different degrees of allergenicity. Methods: Porcine and shrimp tropomyosin (ST) were subjected to sequential exposure to amylase, pepsin, and pancreatin in their respective endogenous matrix (pork tenderloin/boiled shrimp) and in three different experimental matrices (dessert mousse [DM], soy milk [SM], and chocolate bar [CB]). Digestion was monitored by immunoblotting using tropomyosin-specific antibodies. Recombinant peach and strawberry lipid transfer protein were biotinylated, spiked into both peach and strawberry fruit pulp, and subjected to the same sequential digestion protocol. Digestion was monitored by immunoblotting using streptavidin for detection. Results: Chocolate bar, and to a lesser extent SM, had a clear protective effect against pepsin digestion of porcine tropomyosin (PT) and to a lesser extent of ST. Increased resistance was associated with increased protein content. Spiking experiments with bovine serum albumin (BSA) confirmed the protective effect of a protein-rich matrix. The two tropomyosins were both highly resistant to pepsin in their protein-rich and lean native food matrix. Pancreatin digestion remained rapid and complete, independent of the matrix. The fat-rich environment did not transfer protection against pepsin digestion. Spiking of recombinant peach and strawberry lipid transfer proteins into peach and strawberry pulp did not reveal any differential protective effect that could explain differences in allergenicity of both fruits. Conclusions: Protein-rich food matrices delay pepsin digestion by saturating the protease. This effect is most apparent for proteins that are highly pepsin susceptible in solution. The inclusion of food matrices does not help in understanding why some proteins are strong primary sensitizers while homologs are very poor allergens. Although for induction of symptoms in food allergic patients (elicitation), a protein-rich food matrix that may contribute to increased risk, our results indicate that the inclusion of food matrices in the weight-of-evidence approach for estimating the potential risks of novel proteins to become allergens (sensitization), is most likely of very limited value.

Square-wave retarder for polarization computer-generated holography.

An etched calcite square-wave retarder is designed, fabricated, and demonstrated as an illuminator for an interlaced polarization computer-generated hologram (PCGH). The calcite square-wave retarder enables alternating columns of orthogonal linear polarizations to illuminate the interlaced PCGH. Together, these components produce a speckled, tangentially polarized PCGH diffraction pattern with a measured ratio of polarization of 84% and a degree of linear polarization of 0.81. An experimental alignment tolerance analysis is also reported.

Polarization synthesis by computer-generated holography using orthogonally polarized and correlated speckle patterns.

An interlaced polarization computer-generated hologram (PCGH) is designed to produce specific irradiance and polarization states in the image plane. The PCGH produces a tangentially polarized annular pattern with correlated speckle, which is achieved by a novel application of the diffuser optimization method. Alternating columns of orthogonal linear polarizations illuminate an interlaced PCGH, producing a ratio of polarization of 88% measured on a fabricated sample. The demonstrated technique can be applied to designs for arbitrary irradiance and polarization states in the image plane.

The effects of Platelet-rich Plasma on healing of partial thickness burns in a porcine model.

Platelet-rich plasma (PRP) derived from autologous peripheral blood is rich in platelets that release growth factors and cytokines. We determined the effects of topically applied autologous PRP in a partial thickness porcine burn model. Partial thickness burns were created on the backs and flanks of six domestic pigs (24 burns each) using an aluminium bar preheated to 80° C for 20 seconds. After removing the necrotic epidermis, the burns were randomly treated with a topical antibiotic ointment or a single (day 2), double (days 2 and 7), or triple (days 2, 7, and 14) topical application of PRP that was prepared freshly before application. Periodic imaging and full thickness biopsies were conducted to monitor healing over 28 days. The percentage wound reepithelialization at days 11, 14, 18 and 21 did not differ significantly among the groups. By day 28 all wounds were completely (>95%) reepithelialized, and there were no differences among the groups. Time to complete healing (presented as mean, [SD]) did not differ among the groups (antibiotics, 17.1 [3.5]; single PRP, 17.6 [4.0]; double PRP, 18.4 [3.9]; and triple PRP, 17.7 [3.3] days; ANOVA P=0.43). Scar depth (presented as mean, [SD]) in mm at day 28 by treatment group was: antibiotic 5.0 [1.0], single PRP 5.5 [1.1], double PRP 5.4 [1.1], and triple PRP 5.5 [0.6], ANOVA P=0.026. We conclude that PRP results in similar rates of reepithelialization and scar depth to standard topical antibiotics in a partial thickness porcine burn model.

Le plasma riche en plaquettes (PRP), dérivé du sang autologue, permet le relargage de facteurs de croissance et de cytokines. Nous avons étudié l'effet de PRP appliqué localement sur un modèle de brûlure intermédiaire chez le porc. Cette brûlure du dos et des flancs était réalisée au moyen de l'application pendant 20 s d'aluminium chauffé à 80°C sur 4 groupes de 6 porcs. Après ablation de l'escarre, les animaux étaient tirés au sort pour être traités par topique antibiotique, 1 (J2), 2 (J2 et J7) ou 3 (J2, J7, J4) application locale de PRP préparé juste avant utilisation. Des photos et des biopsies ont été réalisées régulièrement pendant 28 j afin de surveiller la cicatrisation. Les pourcentages de surface cicatrisée à J11, J14, J18 et J21 étaient similaires dans tous les groupes. La cicatrisation était quasi complète (> 95%) à J28, dans tous les groupes. Le délai jusqu'à cicatrisation complète n'était pas différent dans les groupes (ANOVA, p=0,43) : Contrôle 17,1 +/- 3,5 ; PRP J2 17,6 +/- 4 ; PRP J2 et J7 18,4 +/- 3,9 ; PRP J4, J7 et J14 17,7 +/- 3,3 jours. La profondeur de la cicatrice, bien que statistiquement significative (ANOVA p = 0,026 ) : 5 +/- 1 (contrôle) ; 5,5 +/- 1,1 (1 PRP) ; 5,4 +/- 1,1 (2 PRP) ; 5,5 +/- 0,6 (3 PRP) mm n'est pas considérée comme cliniquement significative. PRP donne des résultats équivalents aux topiques antibiotiques sur une brûlure intermédiaire du porc.

Immediate tangential excision accelerates wound closure but does not reduce scarring of mid-dermal porcine burns.

Current evidence supports the use of excision to remove eschar from deep dermal and full-thickness burns. However, the role of excision of mid-dermal burns remains unclear. This study aimed to develop a porcine model that could produce reproducible middermal thermal burns that undergo tangential excision; and investigate the effects of immediate tangential excision (30 minutes postburn) on healing and scarring. An aluminum bar preheated in hot water (70°C) was applied for 20 or 30 s to produce a total of sixteen mid-dermal burns per pig on each of six pigs. Thirty minutes after burn creation, half of the burns were tangentially excised. Four partial- thickness wounds per pig were created as controls. Depth of burn injury (1 and 24 h), reepithelialization (7 and 10 d) and scar depth (28 d) were assessed microscopically. Total scar surface area was grossly evaluated on day 28. Exposure of porcine skin to a preheated aluminum bar at 70 °C for 20 or 30 sec resulted in reproducible mid-dermal burns, where immediate excision enhanced complete wound closure as judged by complete re-epithelialization, but did not reduce initial depth of injury, scar contraction and scar depth. Immediate surgical intervention is sufficient to enhance wound closure, but not to mitigate mid-dermal burn scar formation. This work provides a suitable animal model to evaluate novel therapies that may be used to inhibit burn progression, accelerate wound closure and decrease scarring, especially those therapies unable to penetrate burn eschar.

Les données actuelles des connaissances sont en faveur de l'excision des brûlures des 2ème degré profond et 3ème degré. L'intérêt de l'excision des brûlures intermédiaires reste mal précisé. Cette étude se penche sur un modèle porcin destiné à la réalisation de brûlures intermédiaires reproductibles et à l'évaluation de l'effet l'excision ultra précoce (30 mn après la brûlure) sur l'épidermisation et la cicatrisation de ces brûlures. Six porcs ont subi chacun un total de 16 brûlures intermédiaires infligées au moyen d'une barre d'aluminium chauffée à 70°C et appliquée pendant 20 à 30 s. La moitié des zones brûlées étaient excisées à la trentième minute. Quatre brûlures superficielles servaient de contrôle. La profondeur de la brûlure (à h1 et h24), la réépithélialisation (à J7 et J10) et l'épaisseur de la cicatrice (à J28), étaient étudiées microscopiquement. La surface cicatricielle totale était évaluée à J28. L'exposition pendant 20 à 30s de la peau d'un porc à de l'aluminium préalablement chauffé à 70°C entraîne une brûlure intermédiaire reproductible. L'excision immédiate en favorise la guérison lorsqu'elle est jugée sur la réépithélialisation mais n'en réduit ni la profondeur, ni la rétraction cicatricielle, pas plus que l'épaisseur de la cicatrice. L'excision immédiate favorise la fermeture de la plaie mais pas son évolution vers des séquelles. Ce travail permet de décrire un modèle animal fiable dans le but d'évaluer de nouvelles thérapeutiques destinées à limiter le progression des lésions, accélérer la fermeture et diminuer la survenues de séquelles, en particulier celles incapables de pénétrer dans une lésion constituée.

Role of superoxide in the depressed nitric oxide production by the endothelium of genetically hypertensive rats.

We undertook these studies to determine whether a deficient nitric oxide production in genetically hypertensive rats could result from its being scavenged by an excess production of superoxide. In one study we used a porphyrinic microsensor to measure nitric oxide concentrations released by cultured endothelial cells from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY). SHRSP cells released only about one third the concentration of nitric oxide as did WKY cells. Treatment of cells with superoxide dismutase increased nitric oxide release, demonstrating that normally nitric oxide is scavenged by endogenous superoxide. The increase in nitric oxide release in response to superoxide dismutase treatment was more than twice as great from SHRSP as from WKY cells, demonstrating the greater amount of superoxide in the hypertensive rats. A direct measure of superoxide with the use of lucigenin demonstrated the presence of 68.1 +/- 7.1 and 27.4 +/- 3.5 nmol/L of this anion in SHRSP and WKY endothelial cells, respectively. The presence of superoxide in the rat aorta was also estimated by quantification of its effect on carbachol relaxation. This relaxation was diminished when endogenous superoxide dismutase was blocked by diethyldithiocarbamic acid. This blockade reduced the relaxation by 51.2 +/- 5.2% in SHRSP aortas and by only 22.0 +/- 8.2% (P = .015) in WKY aortas. Data from these diverse systems are in agreement that superoxide production is excessive in SHRSP tissues. This excess superoxide, by scavenging endothelial nitric oxide, could contribute to the increased vascular smooth muscle contraction and hence to the elevated total peripheral resistance of these rats.

The glutathione S-transferase M1 genotype in ovarian cancer.

Glutathione S-transferase mu-1 (GSTM1) is a polymorphic member of the mu class gene family of the glutathione S-transferases. Individuals who are GSTM1 null have increased susceptibility to lung and colon cancer. We hypothesized that: (a) GSTM1 null individuals might also be at increased risk for development of ovarian cancer; and (b) the GSTM1 genotype would influence response to chemotherapy. One hundred and forty-six individuals with invasive epithelial ovarian cancer were genotyped using a three-primer PCR reaction specific for the GSTM1 gene and an internal control glutathione S-transferase mu-4 (GSTM4). The products were analyzed on agarose gels. Healthy individuals without a family history of ovarian, breast, or colon cancer served as unmatched controls (n = 80). The results show that age at diagnosis, histological type, and stage of ovarian cancer were all independent of GSTM1 genotype. The frequency of the GSTM1 null genotype in the ovarian cancer cohort was similar to that in the control population, 51% versus 58%, P > 0.05. Likewise, median survival for individuals with advanced stage ovarian cancer was independent of GSTM1 genotype. We concluded that the GSTM1 null genotype does not increase ovarian cancer risk. These findings suggest that GSTM1 does not play a significant role in detoxifying environmental factors that influence ovarian carcinogenesis and does not play an important role in the resistance of ovarian cancer to chemotherapy.

Wound healing in ovariectomized rats: effects of chemically modified tetracycline (CMT-8) and estrogen on matrix metalloproteinases -8, -13 and type I collagen expression.

Cutaneous wound healing is a complex process involving interactions of various cell types. Skin, in addition to certain other organs, is dependent on estrogen; and estrogen-deficiency is associated with impaired wound healing. Wound healing involves the action of collagenolytic matrix metalloproteinases (MMPs). We investigated the expression and localization of collagenolytic MMPs -8 and -13 by collagenase activity assay, Western immunoblot analysis, in situ hybridization and immunohistochemical staining as well as type I collagen by hydroxyproline content analysis and immunohistochemical staining in cutaneous wounds from aged Sham and ovarioectomized (OVX) rats. After wounding, OVX rats were treated with either placebo, chemically modified tetracycline-8 (CMT-8) or estrogen. We found that MMP-8 and MMP-13 mRNA were expressed in wound epithelium of all samples examined as evidenced by in situ hybridization. Type I collagen, which was abundant in all groups examined, was decreased in OVX rats, but was increased by both CMT-8 and estrogen treatments to the level of Sham group. Hydroxyproline analysis revealed similar results. Western blot data showed that all forms of MMP-8 and MMP-13 were clearly reduced in the CMT-8 treated group compared to OVX. Analysis of collagenolytic activity confirmed the decreased collagenolysis in skin wound extracts from CMT-treated rats when compared with skin wound extracts from OVX rats. Our results show for the first time that MMP-8 mRNA and protein are expressed in rat wound epithelium. We further show that CMT-8 and estrogen have a beneficial effect on skin wound healing in OVX rats by increasing the collagen content and reducing the MMP-mediated collagenolysis.

Rapid purification of synthetic oligonucleotides: a convenient alternative to high-performance liquid chromatography and polyacrylamide gel electrophoresis.

A new method has been developed to purify and detritylate milligram amounts of synthetic oligonucleotides. Dimethoxytrityl oligonucleotides from 15 to 100 nucleotides in length are applied in triethylammonium acetate or concentrated ammonium hydroxide to a disposable chromatographic cartridge, the NENSORB PREP Nucleic Acid Purification Cartridge. Salts, failure sequences and synthetic by-products are washed away while the desired, full-length, dimethoxytrityl oligonucleotide remains bound to the cartridge. The trityl group is hydrolyzed from the 5'-end of the oligonucleotide with an acid wash and then the purified oligonucleotide is eluted with 35% methanol. Oligonucleotides are recovered salt-free with purities greater than 95%. NENSORB PREP-purified primers provide superior sequence data compared to similar primers used without purification and equivalent data to primers purified by polyacrylamide gel electrophoresis when used in manual radiometric Sanger sequencing.

Crohn's disease and lichen nitidus: a case report and comparison of common histopathologic features.

We describe a 54-year-old black woman with Crohn's disease, who developed lichen nitidus, the third report of a patient with both diseases. The rarity of these diseases individually and the histopathologic features in common imply that the two diseases are linked. Multinucleated giant cells, a common finding in the lesions of Crohn's disease, are less common in the lesions of lichen nitidus. The presence of multinucleated giant cells in lichen nitidus in all three case reports is distinctly unusual. The infiltrates of Crohn's disease and lichen nitidus contain CD-68-positive macrophages. As such, the subset of lichen nitidus with giant cells should be recognized as a cutaneous manifestation of Crohn's disease.

Evaluation of 11-[3H]-tetrodotoxin use in a heterologous receptor binding assay for PSP toxins.

This report describes the preparative scale production of 11-[3H]-tetrodotoxin (TTX) and its evaluation as a substitute for [3H]-saxitoxin (STX) as the radioligand in a receptor binding assay for paralytic shellfish poisoning (PSP) toxins. Restrictions on the world-wide distribution of [3H]-STX imposed by the international Chemical Weapons Convention served as the primary impetus for this study. We have incorporated on a preparative scale, a nonexchangeable tritium label into the TTX molecule at a specific activity of 12.90 Ci/mmol and recovered material of high radiochemical purity (98%). The resulting 11-[3H]-TTX was found to exhibit site-specific binding characteristics in the receptor assay (dissociation constant(K(d))=4.77+/-1.54nM; maximum binding(B(max))=1. 62+/-0.24pmol/mg of synaptosomal protein). The inhibition constant (K(i)) for the assay was 1.46+/-0.28 nM STX equiv. (n=6), with an estimated detection limit of ca. 2-4 ng STX equiv./ml in a sample extract. Moreover, quantitative comparisons indicated that 11-[3H]-TTX could be used interchangeably with [3H]-STX in the receptor assay for determination of PSP toxicity in shellfish and algal extracts without compromising assay performance. We conclude that the 11-[3H]-TTX produced and evaluated herein exhibits physical, chemical and biological characteristics suitable not only for use in the PSP receptor binding assay, but likely for other applications employing [3H]-STX as the radioligand.

The effects of octylcyanoacrylate on scarring after burns.

OBJECTIVE: To compare the effects of octylcyanoacrylate (OCA), silver sulfadiazine (SSD), polyurethane film (PU), and dry gauze (G) on scarring three months after partial-thickness burns. METHODS: This was a prospective, blinded, controlled experimental trial using isoflurane-anesthetized swine. Standardized partial-thickness burns were inflicted by applying an aluminum bar preheated to 80 degrees C to the backs and flanks of a young pig for 20 seconds. Four equal sets of ten burns each were randomly treated with OCA spray, SSD, PU, or G. Dressing changes were performed on days 1, 2, 3, and 4 after injury. Digital images of the burns were obtained immediately and three months later for masked computerized determination of scar surface area. Full-thickness biopsies were taken at three months for masked histopathological evaluation. The primary outcome was the percent reduction in residual wound area (RWA) calculated by subtracting the area of each individual burn from the area of the largest burn and dividing this value by the area of the largest burn (intraobserver correlation, r = 0.99). Secondary outcomes were the proportion of burns with the presence of scar tissue (abnormal collagen under polarized light; intraobserver agreement, kappa = 0.93) and the cosmetic appearance on a 100-mm visual analog scale marked "best scar" at the high end (inter-observer correlation, r = 0.82). Analysis of variance (ANOVA) and chi(2) tests were used for group comparisons as appropriate. This study had 80% power to detect a 33-percentage-point difference in RWA among groups (alpha = 0.05). RESULTS: A total of 40 burns were inflicted on the pig. There was no difference in percent RWA across the groups (OCA = 25%, SSD = 40%, PU = 25%, G = 32%; p = 0.13). There was no difference in the proportion of wounds with scarring among the groups (OCA = 10%, SSD = 22%, PU = 2%, G = 30%; p = 0.89). There was also no difference in the cosmetic scores among the groups (OCA = 78 mm, SSD = 75 mm, PU = 74 mm, G = 74 mm; p = 0.96). CONCLUSIONS: The effects of OCA spray, SSD, PU, and dry gauze on scarring three months after burns in pigs are similar.

Standardized burn model using a multiparametric histologic analysis of burn depth.

OBJECTIVE: Burn depth and extent determine prognosis and therapy. The current classification into first-, second-, and third-degree burns is crude, making comparisons between studies difficult. The authors standardized a reproducible burn model and a precise histopathologic method for describing burn depth in swine. METHODS: This was a prospective, cross-sectional interventional animal study. Eighteen paired sets of burns were inflicted on the clipped flank skin of two anesthetized domestic pigs with a 2.5 cm by 2.5 cm by 7.5 cm aluminum bar preheated in water to 50 degrees C, 60 degrees C, 70 degrees C, 80 degrees C, 90 degrees C, degrees C or 100 degrees C. The bar was applied for 10, 20, or 30 seconds. Full-thickness skin biopsies were obtained 30 minutes after injury for blinded histopathologic evaluation using hematoxylin and eosin staining. Two dermatopathologists made two sets of measurements and were masked to each other's evaluations. The depth of injury was measured with an ocular microtome for each of five dermal parameters: collagen discoloration, intercollagen basophilic material, endothelial cell necrosis, epithelial cell necrosis, and mesenchymal cell necrosis. The correlation between burn depths of the paired sets of experiments was calculated to assess the reliability of the model. Inter- and intraobserver correlations were calculated to assess the reliability of the scale. Analysis of variance (ANOVA) was used to assess the relation between temperature and exposure times on burn depth. RESULTS: Depth of injury for all five dermal elements was related to temperature and exposure times (ANOVA, p < 0.001 for each). The depth of injury in the paired sets of burns was highly consistent (Pearson correlation, range = 0.88-0.95). Inter- and intraobserver correlations were excellent for all measured elements (range = 0.91-0.97 and 0.95-0.99, respectively). CONCLUSIONS: The authors describe a simple and reproducible animal burn model and histopathologic scale for measuring burn depth that they believe will facilitate standardization and comparison within future burn studies.

The effects of epidermal debridement of partial-thickness burns on infection and reepithelialization in swine.

OBJECTIVE: Early postburn debridement of burn blisters is controversial. This study was conducted to compare rates of infection and reepithelialization in debrided vs nondebrided second-degree burns in swine. METHODS: This was a prospective, blinded, controlled, experimental trial using isoflurane-anesthetized swine. Standardized partial-thickness burns were inflicted by applying an aluminum bar preheated to 80 degrees C to the backs and flanks of two young pigs for 20 seconds. In half of the burns the necrotic epidermis was manually debrided. All burns were randomly treated with octylcyanoacrylate spray (OCA) or dry gauze (C). Full-thickness biopsies were taken at 7, 10, and 14 days for blinded histopathologic evaluation. The primary outcomes were the proportions of infected burns at days 7 and 10 and the proportion of completely reepithelialized burns at day 14. Burns were considered infected in the presence of intradermal neutrophils containing bacteria (intraobserver agreement, K = 1.00). A secondary outcome was the proportion of burns with the presence of scar tissue (abnormal collagen under polarized light; intraobserver correlation, K = 0.93). Chi-square tests were used for group comparisons. This study had 90% power to detect a 40-percentage-point difference in infection rates (alpha = 0.05). RESULTS: A total of 126 biopsies from 42 burns were available for review. Infection rates were higher in the debrided burns both at day 7 (55% vs 4.5%, p < 0.001) and at day 10 (65% vs 9%, p < 0.001) after injury. The proportion of nondebrided burns that were completely reepithelialized was higher at days 10 (68% vs 0%, p < 0.001) and 14 (100% vs 65%, p = 0.003). The presence of scar tissue was more common in debrided burns (75% vs 4.5%, p < 0.001). Burns treated with OCA had fewer infections than controls (4% vs 55%, p < 0.001). Fewer OCA-treated debrided burns were reepithelialized at 14 days than those that were not debrided (30% vs 100%, p = 0.001). CONCLUSIONS: Under the current study conditions, early postburn epidermal debridement of second-degree burns resulted in more infections and slower reepithelialization rates in swine. The effects of early postburn epidermal debridement in humans should be explored.

Development of a histomorphologic scale to quantify cutaneous scars after burns.

OBJECTIVE: Cutaneous wound healing in adults invariably results in scarring; however, there are few scales to quantify the degree of such scarring. The authors developed a histomorphologic scale for quantifying scarring after cutaneous burn injury. METHODS: As part of a randomized trial comparing a variety of burn therapies, 40 partial-thickness burns were created on the backs and flanks of anesthetized pigs and treated with a tissue adhesive, antibiotic ointment, occlusive dressing, or dry gauze. Gross scar appearance was independently assessed by two investigators at 90 days on a 100-mm visual analog scale (VAS) marked "best appearance" at the high end. One of the investigators repeated the observation 30 days later. Full-thickness biopsies were taken 90 days after injury and evaluated histologically by a dermatopathologist for the presence of hyperkeratosis, epidermal hyperplasia, presence and depth of scar (defined as abnormally oriented collagen under polarized light), fibroplasia, vascular proliferation, and absence of adnexa, including hair follicles, apocrine glands, and smooth muscles. One point was assigned for each category in the presence of a normal finding, whereas an abnormal finding was assigned a score of zero. The normal dermis (absence of abnormal collagen) was given a score of 3, while decreasing scores of 2 to 0 were given for progressively deeper scars (i.e., 2 for papillary dermis, 1 for upper half of reticular dermis, and 0 for deep dermal lower half). The total histomorphologic score was derived by adding the scores on the individual items. The score ranges from 0 to 10 from worst scarring to absence of scarring, respectively. A subset of observations was evaluated a second time by one of the observers one month later. Intraobserver reliability of the histomorphologic scale was assessed with Spearman's correlation. Inter- and intraobserver Pearson's correlations for the gross scar VAS were calculated, and the correlation between gross and histomorphologic scores was assessed. RESULTS: Intraobserver correlation for individual histomorphologic categories ranged from 0.19 to 1.00. Intraobserver correlation for the total histologic score was 0.95. Inter- and intraobserver correlations for the gross scar VAS were 0.8 each. Correlation between the histomorphologic scale and the gross scar VAS was 0.38. CONCLUSIONS: A new reliable histomorphologic method for quantifying and scoring cutaneous scars is described together with a reliable scar VAS. However, these two scales are not highly correlated.

Octylcyanoacrylate for the treatment of partial-thickness burns in swine: a randomized, controlled experiment.

OBJECTIVE: To compare re-epithelialization rates of superficial partial-thickness burns treated with octylcyanoacrylate (OCA), silver sulfadiazine (SSD), and dry gauze (controls) in swine. METHODS: This was a prospective, longitudinal, blinded, controlled, experimental trial using anesthetized swine. Sixty-three standardized burns were created by applying an aluminum bar preheated to 80 degrees C for 20 seconds to the flanks of four young pigs. Three equal sets of 21 burns were randomly treated with OCA spray, SSD, or dry gauze (controls). Full-thickness biopsies were taken after 30 minutes and at seven and 14 days for blinded histopathologic evaluation by two dermatopathologists using hematoxylin and eosin staining. The percent of wound re-epithelialization was measured at days 7 and 14, calculated by dividing the length of the regenerated epidermis by the measured width of the biopsy. Analysis of variance (ANOVA) and repeated-measures ANOVA controlling for the individual pig were used for comparisons among groups. This study had 80% power to demonstrate a large between-group difference in percent re-epithelization (alpha = 0.05). RESULTS: There were 63 burns and 126 biopsies. Ten biopsies were excluded for technical reasons. At seven days, there was a significant between-group difference in percent re-epithelialization. Percent re-epithelialization was greatest in the OCA group (65.0%), followed by the SSD group (37.6%), and lowest in the control group (8.8%). At 14 days, all wounds demonstrated near complete re-epithelialization and there was no significant difference in the percent of re-epithelialization among the groups. There was only one wound infection in the OCA group. CONCLUSIONS: Under these study conditions, treatment of partial-thickness burns with OCA spray resulted in a higher percent of re-epithelialization at seven but not 14 days when compared with both SSD and control, with no significant increase in infection rates. Future studies should evaluate the use of OCA for the treatment of burns in humans.

Histopathologic effects of cutaneous tape stripping in pigs.

OBJECTIVE: The stratum corneum (SC) is the major barrier to topical absorption of medications. Skin tape stripping (TS) removes the stratum corneum, allowing more rapid absorption of drugs such as local anesthetics. Prior to evaluating TS in human clinical trials, this study was performed to evaluate its immediate and delayed histopathologic effects in swine. METHODS: This was a prospective, interventional, longitudinal, experimental animal study using two isoflurane-anesthetized young swine. Cellophane tape was applied to the skin of clipped swine flanks and peeled away 10, 15, 20, 25, or 30 times. Each level of tape stripping was performed three times in each pig. Full-thickness biopsies were taken at 30 minutes and two weeks later for blinded histopathologic evaluation by a dermatopathologist using randomly ordered hematoxylin-and-eosin-stained tissue sections and conventional light microscopy. The absolute thickness of the cornified layer was measured and compared with normal (unstripped) cornified layer. RESULTS: Tape stripping up to 30 times produces thinning of the SC without detectable changes in the underlying epidermis and dermis at 30 minutes and 14 days post-stripping. The degree of thinning was proportional to the number of tape applications. Complete recovery of the cornified layer was noted at two weeks. There were no adverse effects, such as infection, scarring, or inflammatory cell infiltrates. CONCLUSIONS: Under the conditions studied, TS not only appears safe, but appears to produce no long-term sequelae.

Octylcyanoacrylate for the treatment of contaminated partial-thickness burns in swine: a randomized controlled experiment.

OBJECTIVE: To compare infection and reepithelialization rates of contaminated second-degree burns treated with octylcyanoacrylate (OCA), silver sulfadiazine (SSD), polyurethane (PU) film, and dry gauze (control; C) in swine. METHODS: Eighty standardized burns were created by applying an aluminum bar preheated to 80 degrees C to the backs and flanks of young pigs for 20 seconds. All burns were immediately contaminated with 0.1 mL of Staphylococcus aureus 10(5)/mL and randomly treated with OCA spray, SSD, PU, or dry gauze (C). Full-thickness biopsies were taken at 3, 7, and 14 days for blinded histopathologic evaluation using hematoxylin and eosin (H&E)-stained slides. Burns were considered infected in the presence of interstitial reticular dermal neutrophils containing bacteria (intraobserver agreement, kappa = 1.00). Quantitative wound cultures were performed on a second day-3 specimen. RESULTS: At day 3, wound infection rates were 30% (OCA), 50% (SSD), 55% (PU), and 50% (C); p = 0.40 (Kruskal-Wallis test). At day 7, infection rates were 35% (OCA), 85% (SSD), 70% (PU), and 65% (C); p = 0.01. Median bacterial counts at day 3 were 6,500 (OCA), 20,000 (SSD), 1,000,000 (PU), and 650,000 (C); p = 0.29. The proportion of completely reepithelialized wounds at day 14 were 75% (OCA), 90% (SSD), 85% (PU), and 90% (C); p = 0.50. CONCLUSIONS: Treatment of contaminated partial-thickness burns with OCA spray resulted in fewer infections at one week than with the other three treatments.